Polyvinyl alcohol (PVA) based covalently bonded stable hydrophilic coating for capillary electrophoresis

ABSTRACT

A coated microcapillary column for high performance electrophoresis is disclosed. A preferred microcapillary includes a fused silica capillary column, the inner surface of the column having an interconnected polymeric coating of a polyvinyl alcohol (PVA) based polymer covalently attached to the column wall by Si--O--Si bonds. The resulting coated microcapillary column has good hydrolytic and pH stability and minimizes electroosmotic flow and interactions between sample components and the capillary wall. Also disclosed are a method of forming a polymeric coating layer for any surface by polymerizing the appropriate organic compounds in an organic solvent and a method of forming a column with a hydrophilic polymeric coating by directly converting an attached hydrophobic polymeric coating material to a hydrophilic polymeric coating material.

This application is a continuation of application Ser. No. 08/379,834,filed Jan. 27, 1995, abandoned.

FIELD OF THE INVENTION

This invention relates to coatings for surfaces, and more particularly,coatings suitable for capillary electrophoresis columns.

BACKGROUND OF THE INVENTION

Capillary electrophoretic separation techniques find wide application inthe biologically related sciences. Molecular species such as peptides,proteins, oligonucleotides, and oligosaccharides are separated bycausing them to migrate in a buffer solution under the influence of anelectric field. The separation is normally carried out in thin-walled,narrow-bore capillary tubes to minimize the evolution of heat duringelectrophoretic separation, which would cause zone deformation.

Among the other mechanisms that can cause zone deformation arenon-uniform electroendosmosis, excess electroosmotic flow, and soluteadsorption to the inner surface of the capillary. However, theseproblems can be minimized or overcome by coating the inner wall of theelectrophoresis tube with various polymeric substances.

In U.S. Pat. No. 4,680,201, Hjerten discloses a method for coating theinner wall of a narrow bore capillary with a monomolecular polymericcoating of polyacrylamide bonded to the capillary wall by means of abifunctional reagent, e.g., γ-methacryloxypropyltrimethoxysilane. Thesecapillaries can be used for free-zone electrophoresis in open tubes.

Novotny et al., U.S. Pat. No. 5,074,982, discloses that the inner wallof silica capillaries used in electrophoretic separations can be coatedwith bifunctional reagent using a Grignard reagent, for hydrolyticstability.

Thermal immobilization of adsorbed polyvinyl alcohol (PVA) as a coatingon fused silica capillary surfaces is described in Gilges et al., Anal.Chem. 66:2038-2046 (1994). These coatings are stable for separationsover a wide range of pH; however, at high buffer pH, the adsorption ofPVA molecules and the suppression of analyte/wall interaction isweakened.

SUMMARY OF THE INVENTION

The present invention generally features coatings suitable for surfacessuch as are found in capillary electrophoresis columns and methods fortheir preparation. A microcapillary column of the invention generallyincludes a microcapillary having an interior cavity and a wall with aninner surface, the inner surface of the wall having an interconnectedpolymeric coating that includes a functional group attached to the innersurface and capable of copolymerizing with an organic compound in anorganic solvent and a polymer of the organic compound copolymerized withthe functional group. The coating can be covalently or non-covalentlyattached to the column wall and can further include an additional layerof coating material. Preferably, the organic compound is a vinyl ester,and most preferably, vinyl acetate, and the attached polymer forming theexposed surface of the coating is a polyvinyl alcohol, the hydroxylgroups of which can be further derivatized in any desired manner. In amost preferred capillary column, the coating material includes apolyvinyl alcohol based polymer covalently attached to the column wallby Si--O--Si bonds.

The method of the invention generally includes providing amicrocapillary, modifying the inner surface of the capillary wall toprovide attached functional groups capable of copolymerizing with anorganic compound in an organic solvent, introducing a solution of theorganic compound in an organic solvent into the interior cavity of themicrocapillary; and causing molecules of the organic compound tocopolymerize with the attached functional groups to form aninterconnected polymeric coating material attached to the inner surfaceof the microcapillary column. Preferably, the attached functional groupsare covalently bonded vinyl groups, the organic compound is a vinylester, and the resulting interconnected polymeric coating material ofpolyvinyl ester is modified in a polymer homologous reaction to producethe desired polyvinyl alcohol coating.

In another aspect, the method of the invention features, in general,forming a column with a hydrophilic polymeric coating by directlyconverting an attached hydrophobic polymeric coating material to ahydrophilic coating material. The resulting hydrophobic polymericcoating can contain acidic, basic or neutral functionalities dependingon the intended use of the column.

The terms "CE column" or "microcapillary column" are meant to include avessel of any shape in which capillary electrophoresis can be carriedout. For example, it is also known to use chips with open groovesmicrofabricated into the surface of the chip for capillaryelectrophoresis.

The coating of the invention creates a new, stable surface, appropriatefor CE columns or general surface modification. The coating is stableover a wide pH range and allows highly efficient grafting and/oradsorption of a variety of additional layers, if desired. As used incapillary electrophoresis, the coating suppresses or controlselectroosmotic flow and prevents adsorption of analytes to the surfaceof the column.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, taken inconjunction with the accompanying drawings, in which:

FIG. 1 shows a cross-sectional view of a coated microcapillary column ofone embodiment of the invention in which an interconnected polyvinylalcohol based polymeric coating is covalently attached to the inner wallof the column by Si--O--Si bonds;

FIG. 2 shows the hydrolysis and condensation of vinyltrimethoxysilane toform a mixture of oligomeric vinylsilanol compounds as carried out inthe surface modification step of one embodiment of the method of theinvention;

FIG. 3 shows attachment and curing of the oligomeric vinylsilanolcompounds of FIG. 2 as carried out in the surface modification step ofone embodiment of the method of the invention;

FIG. 4 shows copolymerization of the monomer vinylacetate with the vinylgroups of the attached oligomeric vinylsilanol compounds of FIG. 3 toform a covalently bonded hydrophobic polymer (polyvinyl acetate),according to one embodiment of the method of the invention;

FIG. 5 shows conversion of the covalently bonded hydrophobic polymer(polyvinyl acetate) into its hydrophilic counterpart (polyvinyl alcoholor PVA);

FIGS. 6-9 show open tube capillary zone electrophoresis of proteins atpH 4.4, 8.8, 6.2, and 10.0, respectively, using a microcapillary columnof the invention;

FIG. 10 shows isoelectric focussing of proteins using a microcapillarycolumn of the invention;

FIG. 11 shows isoelectric focussing of hemoglobin variants using amicrocapillary column of the invention;

FIG. 12 shows open tube capillary zone electrophoresis of ΦX174 digestedwith Hae III, using a microcapillary column of the invention;

FIG. 13 shows polymer network separation of DNA sequencing reactionproducts using a microcapillary column of the invention; and

FIG. 14 shows polymer network separation of poly-dA oligonucleotides ofvarious lengths using a microcapillary column of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention, as applied in capillary electrophoresis, provides a newmethod for obtaining highly stable hydrophilic coated microcapillarycolumns with superior performance for the separation of biopolymers. Asshown in FIG. 1, a preferred microcapillary column of the inventionincludes a fused silica microcapillary 10 having an inner wall 12 and aninterconnected polyvinyl alcohol based polymeric coating 14 covalentlyattached to inner wall 12 by Si--O--Si bonds 16.

A preferred method of the invention generally includes, as a first step,a silanization procedure which modifies the silica surface of thecapillary with highly reactive vinyl silanol oligomers, leaving freevinyl groups as a reactive functionality. Next, a hydrophobic monomer(vinyl acetate) is copolymerized with the anchored vinyl groups in anorganic solvent in the interior cavity of the surface modified, fusedsilica capillary. In the last step, the covalently bonded hydrophobicpolymer (polyvinyl acetate) is converted into its hydrophiliccounterpart (polyvinyl alcohol or PVA). (It is possible that some smallamount of acetate groups may remain.) The preparation procedure for thepolyvinyl alcohol-coated capillaries of the invention is simple,reliable and reproducible. No toxic chemicals are involved. Theresulting hydrophilic coating is chemically and hydrolytically verystable and allows the efficient separation of biopolymers over a wide pHrange.

The following procedure (which is described in more detail in theEXAMPLES) will result in a stable coating of polyvinyl alcohol,covalently attached to the surface of fused silica capillaries:

Surface modification of the fused silica capillary to provide stable,covalently bonded groups for copolymerization with monomers of anorganic compound.

To allow reproducible surface modification, the capillaries are firsttreated with acid to compensate for the different surface quality of theindividual fused-silica capillaries (different manufacturers, differentaging history from batch-to-batch). This procedure results in a moreactive capillary surface as well as a more uniform surface by increasingthe number of exposed, reactive silanol groups. Actual modification ofthe capillary wall is achieved by reacting the surface silanols with amixture of oligomeric vinylsilanol compounds. Referring to FIG. 2, thesecompounds are obtained in solution by reacting vinyltrimethoxysilane 22with a limited amount of 0.1M HCl. The acid cleaves off the protectivemethoxy group and converts the silane into a highly surface-reactivepolysilanol species (vinylsilanetriol) 24. In addition, thevinylsilanetriol condenses to oligomers 26. The limited amount of waterpresent prevents the compounds in the mixture from polymerizing andcross-linking, which would result in precipitation. It is to be notedthat these silanization mixtures are stable for days.

Referring to FIG. 3, the subsequent attachment of oligomers 26 to thecapillary surface 12 (via the condensation of surface silanol groups 28with oligomer silanol groups 30) results in a very stable Si--O--Si bond16 and provides the surface with covalently bonded vinyl groups 32 whichcan copolymerize with vinyl acetate. The use of an olefinic,non-functional silane avoids any hydrolytically unstable functionalgroups in the attached molecule. In addition, due to the thermalstability of the vinylsilane group, a curing of the modified surface canbe performed at high temperature (110° C. for 1 h), which increasesstability of the coupling chemistry.

Polymerization of vinyl acetate monomers within the capillary tube in anorganic solvent.

As shown in FIG. 4, copolymerization of vinyl acetate monomers 34 withthe vinyl groups 32 of the covalently bonded vinylsilanes on thecapillary surface 12 results in covalent bonding of the resultingpolyvinyl acetate 36 to the silica surface. (The dotted lines in therepresentation of polyvinyl acetate 36 in FIG. 4 indicate the earlierlocation of the double bonds in the vinyl acetate monomers. Thoseskilled in the art use the term poly vinyl acetate for the polymer ofvinyl acetate monomers even though the resulting polymer no longercontains vinyl groups.)

Polymerization is initiated in an organic solvent, in the capillarytube, by the thermal decomposition of radical initiators such as α,α'-azodiisobutyronitrile or benzoyl peroxide, which start to decomposebelow the boiling point of the monomer. In appropriate circumstances,the polymerization solutions may be deposited as a thin film on themodified capillary wall. This polymerization procedure is not sensitiveto trace amounts of oxygen, as is, e.g., the polymerization ofacrylamide. Organic solvents such as ethyl acetate produce goodpolymerization yields. Since the viscosity of the resulting polymersolutions in organic solvents is very low, up to 40% monomer can be usedfor the polymerization, guaranteeing a high grafting density(copolymerization with the surface vinyls and thus covalent attachmentof the polymer). Furthermore, excess polymer solution can easily bepushed out of the capillaries. (With acrylamide in aqueous solution, thedensity limits are about 7% monomer.)

Conversion of the covalently bonded hydrophobic polymer (polyvinylacetate) into its hydrophilic counterpart (polyvinyl alcohol or PVA)

After the hydrophobic polyvinyl acetate polymer is bound to thecapillary surface, the coating has to be converted into its desiredhydrophilic form, polyvinyl alcohol (PVA). Referring to FIG. 5, in apolymer homologous reaction, the covalently bonded polyvinyl acetate 36can easily be deacetylated by flushing a solution of sodium methylatethrough the capillaries at room-temperature. Base-catalyzeddeacetylation is very efficient even at ambient temperature; this stepis conveniently carried out by flushing the sodium methylate solutionthrough the capillary for a short period of time--about 15 minutes. Asthe hydrolysis of the ester group is performed under non-aqueousconditions, the silane chemistry is not affected by this strong base.Having anhydrous methanol as a solvent for hydrolysis precipitates theresulting polyvinyl alcohol 14, thus shielding the silane couplingchemistry underneath the polymer. The result of this procedure is amicrocapillary with a hydrophilic, covalently attached coating ofpolyvinyl alcohol, which can be stored until use.

In comparison with, e.g., the polymerization of aqueous solutions ofacrylamide within a capillary, the procedure described above offersseveral advantages. As polymerization is initiated by thermaldecomposition of a radical initiator after the polymerizing solution hasbeen injected into the capillary, the whole procedure is very convenientand controllable. In contrast, conventional polymerization of acrylamidein aqueous solution is initiated as soon as the ingredients (e.g.,TEMED, APS, acrylamide monomers) are combined, and the solution must behandled quickly to prevent premature polymer formation. Polymerizationin organic medium results in much lower viscosities of the polymersolution so that higher monomer concentrations can be used. This resultsin higher grafting density and thus better performance and stability.With 40% vinyl acetate, excess organic solvent can still easily bepushed out of the capillary after polymer formation, while in water, 7%acrylamide is about the limit. Liquid organic compounds that arepolymerizable under the described conditions can, in fact, be usedwithout any additional solvent. In this case, excess compound would bepushed out of the capillary after sufficient polymerization hadoccurred.

Polymerization in an organic solvent is less sensitive to oxygen than ispolymerization in water, which allows the elimination of degassing stepsand permits the use of chemicals as delivered from the manufacturer.This makes the overall procedure much more convenient and gives morereliable results. Finally, the chemicals (vinyl acetate, ethyl acetateand benzoyl peroxide) are much less toxic than those used inconventional, e.g., aqueous acrylamide, polymerization. Use

The above method results in capillaries with excellent performance forthe separation of proteins over a wide range of pH values (see FIGS.6-9). Even at almost neutral pH (pH=6.20, FIG. 8), excellent separationof proteins can be achieved under normal buffer conditions. A capillaryrun constantly at pH 10.0 at a voltage of 540 V/cm showed no loss inefficiency or shift in migration times after 7 days. In contrast, it isknown that all acrylamide- or acrylate-based polymers become chargedupon hydrolysis of functional groups at high pH. This condition resultsin peak deterioration and a shift in migration times for samplesseparated at high pH in polyacrylamide coated CE columns. Additionally,capillaries prepared according to the method of the invention have beenused successfully for isoelectric focusing. Due to their chemicalstability, excellent migration time reproducibility could be achieved.

Any of a variety of other vinyl esters could be used according to themethod of the invention to make a polyvinyl alcohol coating, covalentlybonded to the inner surface of a capillary column. In addition to thepreferred ester, vinyl acetate, other appropriate vinyl esters includevinyl propionate, butyrate, benzoate, or laurate. Furthermore, themethod of the invention is effective in forming a coating layer from anyorganic compound (monomer or oligomer) that is capable of copolymerizingin an organic solvent with functional groups attached to the capillarywall. Moderately hydrophobic monomers (e.g., vinylpyrrolidone,hydroxyalkyl(meth)acrylates, etc.) may be used to produce attachedpolymers that are sufficiently hydrophilic for use for CE separation ofbiopolymers in aqueous buffers without subsequent polymer homologousconversion.

The anchored functional groups can be attached covalently ornon-covalently to the column wall. Functional groups that cancopolymerize with vinyl esters preferably include vinyl groups, but canalso include any other functional group that can copolymerize in anorganic solvent (such as allyl, acryl, methacryl or any otherdouble-bond containing group). Other functional groups would be used forpolymerizing with other polymerizable organic compounds.

The capillary is preferably made of fused silica and the anchoredfunctional groups are preferably covalently attached to the innersurface of the column by the silane coupling chemistry described;however, other coupling methods will be obvious to those skilled in theart. The capillary may also be made of any organic polymer that alreadycontains an appropriate functional group or that allows copolymerizablegroups to be bonded to the surface.

Coating of other surfaces that are of importance in separationtechnologies (such as silica gel, polystyrene, etc.) could easily becarried out using the method of the invention. The preferred methoddescribed herein of applying a PVA coating is applicable to all silicasurfaces (or polymer surfaces) modified with groups that cancopolymerize with the monomer. This procedure would be of particularimportance for HPLC, where surfaces with low protein adsorption arehighly desirable, especially for size exclusion chromatography ofproteins.

Coatings prepared by the method of the invention can easily bederivatized or modified to change surface chemistry or to attach anadditional coating layer. For example, modifications to a covalentlybonded PVA-coating (prepared as described) in a capillary or on thesurface of silica gel can include crosslinking by bi-(or poly)functionalreagents (such as diepoxides, diisocyanates, acid anydrides); chemicalconversion of the polyhydroxy-coating into polyethers, polyesters, etc.,by chemical reaction of the hydroxy-functionality with monomeric orpolymeric reagents; linking of adjacent hydroxy-functionalities as in anepoxide or acetal; or reacting the functional surface with groups thatwould introduce charges and may result in an ion-exchange capacity ofthe surface. Thermal treatments may result in physical (orientation ofthe polymeric layer, hydrogen bonding, partial crystallization) orchemical (partial or complete condensation and thus crosslinking)modifications of the coating. Modification of the polymerizationconditions or chemical conversion after polymerization to affect thedistribution of 1,2- and 1,3-diols is also possible.

Use of borate containing buffers with a PVA coated column could resultin complex formation with PVA hydroxyl groups. The resulting surfacecharge, at high pH, gives an EOF comparable to a bare fused silicacapillary. Thus, PVA coated capillaries may be considered as capillarieswith switchable (buffer dependent) EOF; with PVA coated HPLC-supports,borate buffers could be used to generate a dynamic cation exchanger.

The surface chemistry possible with PVA-coated silica surfaces allowsfor a broad range of chemical reactions as the surface can be consideredin general as a polyhydroxy-compound (polyalcohol). This property isuseful for forming affinity matrices for affinity CE or HPLC asantibodies, or other biospecific reagents, can easily be bonded to thecoated surface via hydroxy-reactive groups. The PVA-coating beneath theattached antibodies would guarantee low adsorption and thus eliminatenon-specific interaction.

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These examples are not intended in any way otherwise tolimit the scope of the disclosure.

EXAMPLE I

Preparation of a covalently bonded polyvinyl alcohol (PVA) coatedcapillary:

A) A length of about 60 cm of polyimide-coated fused silica capillarywith an internal diameter of 75 μm is rinsed for 2 h with conc. HCl/H₂ O(1:1) at a temperature of 110° C. to provide a surface with high silanolconcentration for successful silane coupling chemistry. The capillary isthen rinsed to neutral pH and dried in a gentle He stream.

B) The silanization solution is prepared by mixing 2 ml ofvinyltrimethoxysilane and 0.4 ml of 0.1M HCl. Upon stirring, the initialtwo phase mixture homogenizes due to hydrolysis of alkoxysilane groupsand liberation of methanol, a very exothermic reaction. One hour isallowed for hydrolysis and condensation of the vinylsilanols. Thesolution of oligomeric vinylsilanols is pushed through the capillariesfor 1 h and allowed to stand overnight. The next day the capillaries arerinsed with methanol. Curing is performed at 110° C. in a gentle Hestream.

C) The polymerization solution is prepared by mixing 3 ml of ethylacetate with 2 ml of vinyl acetate, resulting a concentration of vinylacetate of 40% (V/V). The addition of 100 μl of a 5% solution of benzoylperoxide in ethyl acetate yields an initiator concentration of 0.25%.The vinyl modified capillaries from B) are filled with thepolymerization solution and the polymerization is performed at 75° C.for 20 h. The polymer solution is then pushed out, and the capillariesare rinsed with ethyl acetate and methanol to remove any non-bondedpolyvinyl acetate.

D) To convert the covalently bonded polyvinyl acetate into polyvinylalcohol, the coated capillaries from C) are rinsed for 15 min with asolution of 0.5M sodium methylate in methanol. The capillaries arerinsed afterwards with methanol and then dried in a He stream.

EXAMPLE II (FIG. 6)

Sample: (1) lysozyme, (2) cytochrome C, (3) myoglobin, (4) trypsinogen,(5) α-chymotrypsinogen A (0.1 mg/ml each). Coating: PVA as described inExample I. Conditions: i.d.=75 μm; L=30/37 cm; buffer: 20 mMε-aminocaproic acid, pH=4.40; injection: 4 sec at 5 kV; separationvoltage: 20 kV.

EXAMPLE III (FIG. 7)

Sample: (1) glucose-6-phosphatedehydrogenase, (2) trypsin inhibitor, (3)L-asparaginase, (4) α-lactalbumin (0.1 mg/ml each). Coating: Example I.Conditions: i.d.=75 μm; L=30/37 cm; buffer: 20 mM TAPS/AMPD, pH=8.80;injection: 4 sec at 5 kV; separation voltage: 20 kV.

EXAMPLE IV (FIG. 8)

Sample: (1) lysozyme, (2) cytochrome C, (3) myoglobin, (4) trypsinogen,(5) α-chymotrypsinogen A (0.1 mg/ml each). Coating: Example I.Conditions: i.d.=75 μm; L=30/37 cm; buffer: 20 mM TRIS/cacodylic acid,pH=6.20; injection: 5 sec with pressure injection (PAC/E); separationvoltage: 20 kV.

EXAMPLE V (FIG. 9)

Sample: (1) glucose-6-phosphatedehydrogenase, (2) trypsin inhibitor, (3)L-asparaginase, (4) α-lactalbumin (0.1 mg/ml each). Coating: Example I.Conditions: i.d.=75 μm; L=30/37 cm; buffer: 20 mM CAPS/NaOH, pH=10.0;injection: 4 sec at 5 kV; separation voltage: 20 kV.

EXAMPLE VI (FIG. 10)

Sample: (A) myoglobin (pl=7.2), (B) carbonic anhydrase 1 (pl =6.6), (C)carbonic anhydrase II (pl=5.9), (D) β-lactoglobulin A(pl=5.1) (0.1 mg/mlof each protein mixed 1:1 with 2% Pharmalyte (3-10)). Coating: ExampleI. Conditions: i.d.=50 μm; L=30/37 cm; focussing: at 25 kV;mobilization: low pressure (PAC/E) starting after 10 min (25 kV);anolyte: 20 mM H₃ PO4; catholyte: 20 mM NaOH.

EXAMPLE VII (FIG. 11)

Sample: hemoglobin variants C (pl=7.45), S (pl=7.20), F (pl=7.00) and A(pl=6.95) (0.1 mg/ml each mixed with 1:1 with a 2% ampholine mixture(Pharmalyte, Servalyte and Ampholyte)). Coating: Example I. Conditions:i.d.=50 μm; L=30/40 cm; focussing: at 30 kV; mobilization: hydrodynamic(d H=5 cm) starting after 15 min (30 kV); anolyte: 0.5% acetic acid;catholyte: 0.25% ammonium hydroxide.

EXAMPLE VIII (FIG. 12)

Sample: ΦX174 digested with Hae III. Coating: Example I. Conditions:i.d.=100 μm; L=26.75/27.50 cm; sieving matrix: 1% methylcellulose (2%gives 4000 cps) in 40 mM TAPS/TRIS; injection: 5 sec at 5 kV; separationvoltage: 5 kV.

EXAMPLE IX (FIG. 13)

Sample: FAM labeled primer sequencing reaction terminated withdideoxythymidinetriphosphate on M13 mp18. Coating: Example I.Conditions: i.d.=100 μm; L=30/40 cm; sieving matrix: 4%T LPA in 40 mMTRIS/TAPS with 30% formamide and 3.5M urea; separation voltage: 8 kV;injection: 5 sec at 8 kV.

EXAMPLE X (FIG. 14)

Sample: poly-da oligonucleotides 12-8, 25-30 and 40-60. Coating: ExampleI. Conditions: i.d.=100 μm; L=20/27 cm; sieving matrix: 10%polyamide+45% DMSO+10% urea+35% TAPS/TRIS (50 mM); injection: 3 sec at10 kV; separation voltage: 20 kV; temperature: 40° C.

EXAMPLE XI

Coating a stationary phase for use in HPLC with polyvinyl alcohol. Asilica gel of 300 Å pore size and 5 μm particle diameter is modifiedwith vinyl groups as described in Example I (B) for a fused silicasurface or according to other procedures well known to those skilled inthe art (E. P. Plueddemann, Silane Coupling Agents. Plenum, N.Y. (1982))5 g of the silica gel is suspended in a solution of 10 ml vinyl acetateand 30 ml ethyl acetate. After 25 mg of benzoyl peroxide is added, thesuspension is heated up to 75°0 C. for 20 hours under stirring andreflux. The polymer solution is then removed with a G4 filter funnel,and the polyvinyl acetate coated silica gel is washed with ethyl acetateand methanol to remove any non-covalently adsorbed polymer. Homologousconversion of the bonded polyvinyl acetate into PVA is carried out bystirring the silica gel for 15 min in a 0.1M solution of sodiummethylate in methanol. Then the silica gel is washed with methanol anddried at a temperature of 60° C.

EXAMPLE XII

The PVA-coated silica gel of Example XI is packed into a HPLC column,and high performance (or high pressure) size exclusion chromatography(SEC) of proteins, nucleotides and synthetic polymers can be performedin organic or aqueous solvents.

EXAMPLE XIII

Preparation of affinity matrices from PVA-coated surfaces can beprepared according to procedures well known to those skilled in the art,as disclosed in, e.g., G. T. Hermanson, A. K. Mallia, P. K. Smith,Immobilized Affinity Ligand Techniques. Academic Press, San Diego(1992).

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of this specification or practice of theinvention as disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A coated microcapillary column for high precisionand high performance electrophoresis, comprising:a microcapillary columnhaving an interior cavity and a wall with an inner surface, said innersurface of said wall having an interconnected, polymeric,surface-modifying coating comprising a polyvinyl alcohol based polymercovalently attached to said inner surface of said wall, said coatingremaining stable and covalently attached to said wall at pH valuescomprising pH 4.4 to pH 10.0.
 2. The coated microcapillary column ofclaim 1 wherein said microcapillary is made of a material selected fromthe group consisting of fused silica, glass, polytetrafluorethylene, andpolyether ether ketone.
 3. The coated microcapillary column of claim 1wherein said coating is further modified.
 4. The coated microcapillarycolumn of claim 1 wherein said coating further comprises an additionallayer of coating material.
 5. The coated microcapillary column of claim4 wherein said additional layer of coating material is covalently bondedto hydroxyl groups of said polyvinyl alcohol based polymer.
 6. Thecoated microcapillary column of claim 1 wherein, in said coating, saidpolyvinyl alcohol based polymer comprises free hydroxyl groups.
 7. Thecoated microcapillary column of claim 1 wherein, in said coating, saidpolyvinyl alcohol based polymer comprises derivatized hydroxyl groups.8. The coated microcapillary column of claim 7 wherein said derivatizedhydroxyl groups of said polyvinyl alcohol based polymer comprisehydroxyl groups on adjacent carbon atoms derivatized to form an epoxideor an acetal.
 9. The coated microcapillary column of claim 1 wherein, insaid coating, said polyvinyl alcohol based polymer is covalentlyattached to said inner surface of said wall by Si--O--Si bonds.